Co-culture Wood Block Decay Test with Bacteria and Wood Rotting Fungi to Analyse Synergism/Antagonism during Wood Degradation

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments. Key features • Quantification of wood decay ability of mixed cultures. • Allows testing if fungi are more efficient in degrading wood when bacteria are present.

Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany).Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability.Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains.Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments.This protocol provides new opportunities in exploration of inter-and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments.

Background
In nature, basidiomycetous fungi are associated with other microbes including prokaryotes and eukaryotes.This results in complex competitive and antagonistic interactions as well as commensal and mutualistic behaviour (Boer et al., 2005;Kobayashi and Crouch, 2009).Fungi are the most efficient wood decomposers, as their multicellular appearance and hyphal growth harbour a mobility advantage in comparison to prokaryotes.Nonetheless, bacteria are known to have direct influence on the decay process as well, by e.g., degrading complex wood components like cellulose, lignin, and hemicellulose (McGuire and Treseder, 2010) or by altering wood permeability and structure, thus improving accessibility of the wooden microfibrils (Clausen, 1996), aiding other organisms in wood decay.Synergistically acting species benefit from each other's enzymatic abilities when they are cultivated on wood (Cortes-Tolalpa et al., 2017; Sugano et al., 2021).These results highlight the importance of inter-and intra-kingdom interactions, as they show that the combined action of different species enhances the decay ability of the whole community.
To date, several protocols and studies exist for the assessment of wood resistance against wood-destroying basidiomycetes [e.g., DIN V ENV 12038:2002 (German Institute for Standardisation, 2002), wood block test of Bravery (1978), or work of Lohwag (1965) and Hegarty et al. (1987)].However, as these protocols evolved in large part to study timber preservation and were not designed to study the influence of other microbes, we adapted a protocol based on DIN V ENV 12038:2002 to the here described co-culture wood block decay test.This step-bystep protocol provides an easily feasible standard procedure for the assessment of wood decay properties of mixed cultures in comparison to axenic decay rates by evaluating the dry weight of wood blocks that were exposed to microbial deterioration.The purpose of this wood decay assay is to determine the influence of bacteria on the decay properties of wood rotting fungi like Serpula lacrymans [see e.

B. Preparation of the main experiment
1. Establish fungus on solid agar medium (Figure 1; here, S. lacrymans was pre-cultivated on modified Melin-Norkrans (MMN) for 3-4 weeks at 25 °C).Note: Use the peripheral zone of the pre-culture(s).Set aside some vessels, as they serve as control (Figure 1).Note: Set aside some vessels as they serve as control.6.For each approach, put wood blocks in vessels filled with MEA but without mycelium, as these are additional controls (axenic bacterial controls) (minimum triplicates per bacterium).7. Treat a minimum of 14 wood blocks solely with 0.85% NaCl solution and place them on top of the mycelium (axenic fungal control).8. Incubate the vessels at 25 °C in darkness (e.g., put them in a carton package) for eight weeks (Figure 2).a.For plate casting after Koch (if less than 1 mL): i. Mix residual solution with 3 mL of TSA soft agar.
iii.Pour evenly on TSA plates.b.For concentration on a nitrocellulose filter with vacuum (if more than 1 mL): i. Clean the filter unit with 96% EtOH and sterilise the tweezers.
ii. Install the sterile filtration funnel and the filter.
iii.Pour the solution on the filter.iv.Apply vacuum.v. Put the filter on TSA medium.8. Incubate all agar plates at 25 °C and check daily for growth (Figure 3).2) Put it on the thermo shaker (37 °C, 800 rpm) for 15 min.
3) Centrifuge at 15,000× g for 15 min at RT (21 °C).4) Discharge the supernatant (as it contains leftover polymerases, primer dimers, and unused dNTPs interfering with the subsequent sequencing reaction).5) Wash the pellet with 50 μL of ice-cold 80% ethanol (-20 °C).6) Centrifuge at 15,000× g for 2 min at RT (21 °C).7) Remove the supernatant.8) Repeat the centrifugation step and remove the supernatant completely.9) Incubate the tubes on the thermo shaker (37 °C and 800 rpm) with lid open until no trace (visible drops as well as smell) of ethanol is left.10) Add 18 μL of nuclease-free water.11) Pipette the nuclease-free water up and down several times (this ensures that the DNA is fully resuspended in the water).vi.Check the purified PCR products again with agarose gel electrophoresis (sodium borate buffer 1×, 100 V, approximately 20 min) (Figure 3).vii.Send for sequencing [using e.g., the Microsynth sequencing service (Balgach, Switzerland)].

A. Wood decay rates
To ensure that the initial moisture content was uniform throughout all wood blocks and to calculate the estimated dry weight on day zero (dry m0), dry four moisture control specimen wood blocks at ~50 °C for 72 h after autoclaving and weighing (point B5).This can be done in parallel to the assay.Our specimen lost on average 0.6905 g (≙8.62%), hence 91.38% of the mass remained unchanged.Calculate dry m0 as: dry m0 = m d0 × 0.9138, with m d0 being the initial weight of the wood block after autoclaving (before exposure to microbes).Calculate mass loss (ML) difference as: mT (g) = (dry m0 -m1), where mT is the difference of ML after exposure to microorganisms, and m1 and dry m0 are the dry masses after and before degradation, respectively.The ML in % is the measure for the extent of fungal degradation [mT (%) = (dry m0 ÷ m1) × 100].Prepare a minimum of five replicates for each approach and of three replicates per control approach.Calculate average wood weight loss in % and standard deviation for each approach and illustrate e.g., in a boxplot.Conduct statistical analysis with pairwise.t.test function for pairwise comparison using t-tests with pooled SD (p-value adjustment method: holm and Bonferroni) in the psych package v2.2.5 of the R programming language (RStudio).Additionally, calculate single-factor variance analysis [ANOVA, function aov() in RStudio].A significantly higher weight loss in the co-cultivation approach than in the axenic fungal control is interpreted as a synergistic effect; lower weight losses are interpreted as antagonism.

B. Evaluation of PCR 16S rRNA placement
Check sequencing results for quality by evaluating the corresponding chromatograms.If 16S rRNA gene sequences of the applied microbes are available for comparison, compare initial sequences with the sequences of the re-isolated strains by aligning them.Alternatively, blast sequences of the 16S rRNA gene with the NCBI online tool.Limit the searching settings to 16S ribosomal RNA sequences (Bacteria and Archaea) and optimise for highly similar sequences (megablast).According to the results of the Blast search, assign the bacteria to genera and compare with the bacterial strains applied at the beginning.This allows to estimate if the bacteria survived the procedure.
Note: Some mould spores may have survived and developed; we recommend recording affected plates/tins.We excluded these specimens from the calculations.

Notes
Prepare enough tins with pre-cultured fungus, as contaminations occur easily since some steps cannot be performed entirely sterile.Before starting the experiment, we recommend careful planning and listing of what should be prepared.

Mind the controls:
• Axenic fungal control: S. lacrymans with wood that is immersed with 0.85% NaCl solution.
• Wood sterile control: wood that is immersed with 0.85% NaCl solution, without microbes.
• Axenic bacterial control: wood that is immersed with bacterial suspension that was adjusted to 10 8 CFU/mL without fungus.• Minimum four moisture control specimen wood blocks (without them it is not possible to estimate the initial dry weight dry m0).It is also possible to sterilise the wood blocks via UV light, as this would preserve the initial state of the wood better than autoclaving.The disadvantage is that more moulds and other contaminants survive this procedure, which might cause problems in the experiment.

Published: Oct 05, 2023 FeSO4• 7H2O, 4 .
99 g in 50 mL 20% KOH solution (w/v) For 1 L final mix, dissolve each component in the volume of water indicated.The EDTA should be dissolved in boiling water, and the FeSO4• 7H2O should be prepared last to avoid oxidation.Mix all solutions except EDTA.Bring to boil, then add the EDTA solution.The colour of the mixture turns to green.When everything is dissolved, let cool to 70 °C.While keeping the temperature at 70 °C, add 85 mL of hot KOH (20%).Cool to RT and fill up to 1 L final volume.Close the flask with a cotton plug (allows air exchange) and swirl it once a day while incubating for 1-2 weeks.Usually, the solution will initially be clear green but turns dark red or purple over the next few days, leaving a rust-brown precipitate.If no precipitate forms or the solution remains green, check the pH (should be at approximately 6.7; if there is a big deviation, try adding either KOH or HCl to adjust it).Filter through two layers of Whatman #1 filter paper and repeat, if necessary, until the solution is clear.Store refrigerated or frozen in convenient aliquots.

ProcedureA.
Pre-experiments to determine bacterial viability on wood blocks1.Grow bacterial isolates in 5 mL of TS liquid medium overnight (25 °C, 220 rpm, uncontrolled light/dark conditions).2. Harvest bacteria and wash them with 1 mL of 0.85% NaCl solution (3×, centrifugation at 5,900× g for 10 min).3. Use a Thoma chamber to adjust the bacterial suspension to 10 8 CFU/mL with 0.85% NaCl solution (end volume minimum 10 mL). 4. Immerse the autoclaved wood blocks with bacterial suspension and incubate them in a sterile glass Petri dish overnight at 25 5.The next day, imprint the wood blocks gently on TSA plates, using a sterilised tweezer.Remove the wood blocks.Incubate at 25 °C overnight to 24 h.6.If bacteria grow rapidly on TSA plates, they are useable for the main experiment.

3 .
Incubate in darkness for four weeks (25 °C).In the meantime: 4. Number the wood blocks (50 ± 0.5 mm × 25 ± 0.5 mm × 15 ± 0.5 mm, e.g., from Picea abies).5. Autoclave wood blocks (120 °C) and note their weights (maintain sterile conditions, e.g., put balance under the sterile workbench).Keep them in a sterile environment until use.a.Put a minimum of four moisture control specimen wood blocks in the oven and dry them at ~50 °C for 72 h.b.Weigh and record the dry mass of the moisture control wood blocks.6. Number and weigh glass Petri dishes.C. Setup of the main experiment 1. Start to prepare bacteria approximately 2-3 days before fungal cultures are ready.Culture them on TSA medium (25 °C). 2. Inoculate 5 mL of liquid TS medium with bacteria (25 °C, 220 rpm, overnight).3. Harvest bacteria and wash them with 0.85% NaCl solution (3×, centrifugation at 5,900× g for 10 min) 4. Use a Thoma chamber to adjust the bacterial suspension to 10 8 CFU/mL (use 0.85% NaCl solution).5. Immerse the numbered, autoclaved, and weighed wood blocks with the bacterial suspension, remove them immediately, drain the excess suspension (sterilised filter paper) and put one (immersed) wood block in each vessel with fungal mycelium.Prepare a minimum of five replicates.

Figure 2 .
Figure 2. Preparation and setup procedure of the wood decay experiment.Illustration of sections B and C.

Figure 3 . 1 .
Figure 3. Harvesting and evaluation procedure of the wood-decay experiment.Illustration of sections D and E.